Disposable plate electrode with biologicalactive film and manufacture method thereof

ABSTRACT

A manufacture method for forming a disposable plate electrode with biological active film is used to cooperate with a biological sensor for analyzing composition and measuring concentration of a test sample according to electric effect resulted from a biochemical reaction. The plate electrode comprises at least an electrode portion for transmission of the electric effect as well as a biological active film that reacts with the test sample chemically or biochemically. The biological active film contains a carrier layer (cellulose, for example) for adsorbing and keeping the biological active substance (enzyme, for example), which, the carrier layer, can change the electrode portion from hydrophobic into hydrophilic and protect the biological active substance against impairment during relatively higher temperature drying process. The method for forming a biological active film on the disposable electrode is mainly based on screen printing technique to form a conductive film, an electric insulating layer, a carrier layer, etc, for speedy production and low cost purpose.

BACKGROUND OF THE INVENTION

[0001] This invention relates to a disposable plate electrode withbiological active film and manufacture method thereof, particularly to amanufacture method that can produce plate electrode speedily and to adisposable plate electrode which is used to cooperate with a biosensorfor composition analysis and concentration measurement of a test samplein accordance with the electrical output resulted from biochemicalreaction.

[0002] The biochemistry analyzer is usually classified into threecategories, namely, the wet type analyzer, the dry type analyzer, andthe biosensor. Application of a conventional wet type biochemistryanalyzer is to mix a test sample with reagent (a chromatic agent iscommonly contained) for chemical reaction, then an optical readingdevice, such as a colorimeter or a spectrophotometer, is used to readcolor change before and after the reaction. This test way is weak at: apre-treatment required for the test sample; difficulties in dosing andkeeping a reagent valid for a long period; expensive instruments; andunfeasibility for non-professional operation, so that it fits a hospitalor an examining center for mass sample analyses better than few quantityor emergency tests.

[0003] As to application of the dry type biochemistry analyzer, a teststrip is coated on its surface with a chemical reagent, such as anenzyme, or antibody, etc to contact directly and react with the testsample for analysis. Though this test way can save dosing and addingprocess of the reagent, color of the test strip may be changed due tooxidation to affect color judgment before and after reaction.

[0004] The biosensor is composed of a biological element, a thin filmelement, and a sensor, wherein the biological element is made frombiological material with cognizable specialties, such as microbe, cell,tissue, enzyme, antigen, and antibody, etc; the thin film element isusually made from polymeric material and used to fix the biologicalelement and sieve out interference substance; and, the sensor maycomprise electrodes, ion selective field effect transistors,thermistors, piezoelectric devices, optical fiber, photoelectric tubes,and sound wave counters, etc., and wherein the hydrogen peroxideelectrode is the one most widely used.

[0005] Take the biosensor for analyzing blood glucose for instance,glucose is oxidized and fixed on a thin film, which is clad tightly onsurface of a pillar hydrogen peroxide electrode, then a polarizedpotential is applied to the platinum anode and the silver/silverchloride cathode, the hydrogen peroxide produced by oxidation of theglucose will continue to be oxidized to water near surface of the anode,and meanwhile, release electrons. The glucose concentration of the testsample may be calculated according to the released amount of electrons.

[0006] The aforesaid pillar electrodes shall require constantrefreshment including polishing, film clothing, cleaning, andrecalibrating, etc, which may incur cross pollution owing tocarelessness in addition to inconvenient implementation, not to mentionthe high production cost. For eliminating above defects, the U.S. Pat.No. 4,545,382 of Genetics International in UK has disclosed a bloodglucose meter “Exactech” which is the first commercially realizedexample of a plate electrode in this field.

[0007] In U.S. Pat. No. 5,120,420—Biosensor and process for preparationthereof, another disclosed biological detecting plate electrodecomprises an electrode portion, an insulation layer, a reaction layer,and a test sample bearing space on the reaction layer, wherein a sampleinlet port and an gas exhaust port are provided to the bearing space.The reaction layer is formed by coating subsequently CMC (CarboxymethylCellulose) water solution on the electrode substrate to form a CMClayer, water solution of GOD (Glucose Oxidase), and then a suspendingliquid containing conductive mediator to form a biochemistry reactionportion. Finally, a resin plate and a top plate are used to cover on thesubstrate to form the sample bearing space and complete thereby thebiological detecting plate electrode.

[0008] The biochemistry reaction portion of the aforesaid U.S. Pat. No.5,120,420 is formed in 3 steps, namely:

[0009] 1. The CMC layer used to improve hydrophobicity of the carbonelectrodes.

[0010] 2. The GOD layer.

[0011] 3. The conductive mediator layer.

[0012] Each step requires drying before completed.

SUMMARY OF THE INVENTION

[0013] This invention is proposed to provide a new fabrication processfor plate electrode, which is provide at least with an electrode portionused to transmit electrical effect produced from biological reaction;and a biological active layer used to conduct a chemical or biochemicalreaction with a test sample. The biological active layer comprises atleast 3 portions: an absorptive carrier layer, an enzyme, and aconductive mediator. The carrier is printed on surface of the electrodeportion with screen-printing technique and used to suck and sustain thebiological active substance (such as enzyme) and the conductivemediator. Also, the carrier can turn the electrode portion fromhydrophobic into hydrophilic, and moreover, protect the biologicalactive substance against impairment in relatively high temperaturedrying process to make a speedy production of the plate electrode becomepossible.

[0014] Another object of this invention is to provide a simplifiedprocess for production of the plate electrode, wherein the carrier layeris printed on an insulating substrate of the plate electrode withscreen-printing technique.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a better understanding to the present invention, togetherwith further advantages or features thereof, at least one preferredembodiment will be elucidated below with reference to the annexeddrawings in which:

[0016]FIG. 1 is an elevational view showing structure of a plateelectrode of this invention;

[0017]FIG. 2 is a front view showing structure of the plate electrode ofthis invention;

[0018]FIG. 3 is a cutaway sectional structure along line III-III in FIG.2 of the plate electrode of this invention;

[0019]FIGS. 4A through 4H show production flow charts of this inventionin preparation steps.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0020] As shown in FIG. 1 and FIG. 2, an approximate strip type plateelectrode is advantageous for loading a test sample 6 and suitable foruse in the following manufacture method. The plate electrode, accordingto the cutaway sectional view in FIG. 3, comprises: a strip substrate 1with plane surface, a conductive film 2 located on one surface of thesubstrate 1, an electric insulating layer 3 locally covering theconductive film 2, and, a biological active layer 4 covering the bareportion of the conductive film 2, which, the biological active layer 4,doesn't overlap the electric insulating layer 3 thoroughly.

[0021] In addition to the plane surface, the foregoing substrate 1 mustbe meanwhile an electric insulator and a heat resister capable ofenduring 40° C.˜120° C. heat treatment in order to increase conductivityand adherence of the conductive film 2. The applicable materials for thesubstrate 1 include PVC (Polyvinyl Chloride), FRP (Fiber ReinforcedPlastics, FR-4), Polyester sulfone, Bakelite, PET, PC, glass, orceramics (CEM-1), etc.

[0022] The conductive film 2 comprises at least a pair of separatedelectrodes, namely, an anode 20 and a cathode 21 to connect with asensor. One of two bare ends of the anode 20 is a work electrode 200covered with the biological active layer 4, and the other is an anodecoupling 201, wherein the work electrode 200 is used to detect theelectric effect induced by chemical or biochemical reaction of the testsample 6, and the anode coupling 201 is used to connect with the sensoras mentioned. As to two bare ends of the cathode 21, one end is areference electrode 210 covered with the biological active layer 4, andthe other is a cathode coupling 211 in arbitrary shape. The referenceelectrode 210 is used to cooperate with the work electrode 200 fordetecting electric effect of the test sample 6, while the cathodecoupling 211 is used to connect with the sensor.

[0023] The electric insulating layer 3 is coated on the same surface ofthe substrate 1 at 0.6 mm thick or up without covering the anodecoupling 201, the cathode coupling 211, the work electrode 200, and thereference electrode 210 to form a circular area on the work electrode200 and the reference electrode 210, namely, the space of biologicalactive layer 4, for placing a biological active substance or the testsample 6.

[0024] This invention is characterized by providing a distinguishingbiological active layer 4 in order to simplify production process of theplate electrode and also to raise its quality. The biological activelayer 4 comprises: a carrier 5 printed on the work electrode 200 and thereference electrode 210 by screen-printing technique, a net protector 7shading on the work electrode 200 and the reference electrode 210, aswell as the biological active substance adsorbed by the carrier 5, andthe conductive mediator.

[0025] The aforesaid carrier 5 is a blended paste for screen-printing,and the ingredients thereof may include:

[0026] 1. Microcrystalline cellulose with grain size at 100 μm down,10˜40% adulterated.

[0027] 2. High molecule polymer with 10˜25% adulterated, such as PVA(Polyvinyl alcohol), PVP (Polyvinyl pyrrolidone), PEG (Polyethyleneglycol), or gelatin. Each single item or blended may be used.

[0028] 3. Salt adulterated at 1˜5%, such as Dibasic potassium phosphate,Potassium biphosphate, and Citric acid. The salt is used to adjust pHvalue and serve as a buffer solution. The proper range of pH value is4.5˜9.0.

[0029] 4. Water. Pure water undergone at least one time distillationwould be a must. Embodiment 1: Microcrystalline cellulose (grain size20˜100 μm) 20% PVA 3.5% PVP 2.8% PEG 12% Gelatin 2.1% Dibasic potassiumphosphate 0.7% Citric acid 1.5% Water 57.4%

[0030] Embodiment 2: Microcrystalline cellulose (grain size 20 μm down)35% PVA 13% PVP 7% Dibasic potassium phosphate 0.7% Citric acid 1.5%Water 42.8%

[0031] Embodiment 3: Microcrystalline cellulose (grain size 20 μm inaverage) 21.2% PEG 19.8% Dibasic potassium phosphate 0.7% Citric acid1.5% Water 56.8%

[0032] Embodiment 4: Microcrystalline cellulose (grain size 20 μm inaverage) 21.2% PVP 13.4% PEG 0.3% Dibasic potassium phosphate 0.04%Potassium biphosphate 0.1% Water 64.96%

[0033] The carrier 5 is coated on surface of the circular area of thework electrode 200 and the reference electrode 210 for adsorbing abiological active substance and conductive mediator to change the carbonelectrodes from hydrophobic into hydrophilic for strengtheningadsorption of the test sample and to assure the biological activesubstance will not be impaired in relatively high temperature dryingprocess, so that the yield of the plate electrode may be raised up. Thebiological active substance means immobilized or unimmobilized enzyme(such as Glucose Oxidase), antigen, antibody, microbe cell, and cell ortissue of animals or plants, which possess biologically discriminativeconstituents, for use to react with a test sample (biological tissuesuch as blood) chemically or biochemically. The conductive mediator(such as potassium ferricyanide, quinones) at content ratio 2˜10% isused to receive the electrons released from the reaction between anenzyme and a test sample, which, the electrons, will be transmitted tothe sensor via a conductor to transfer into concentration of the testsample.

[0034] The biological active substance must be mixed with the conductivemediator, the composition is listed as the following:

[0035] 1. Enzyme, such as Glucose Oxidase with dosage 200 U˜1200 U/ml.

[0036] 2. Enzyme protector with dosage 0.1˜1%, including:

[0037] albumin, dextrin, dextran, or amino acid, which can be usedindependently, or in blended.

[0038] 3. Conductive mediator with dosage 2˜10%, such as Potassiumferricyanide.

[0039] 4. Phosphate buffer solution at pH 4.8˜7.5. Embodiment 5: GlucoseOxidase 0.63% Albumin 0.5% Potassium ferricyanide 6% Phosphate buffersolution at pH 5.0 92.87%

[0040] Embodiment 6: Glucose Oxidase 0.45% Albumin 0.5% Dextran 0.01%Potassium ferricyanide 4.8% Phosphate buffer solution at pH 7.4 94.24%

[0041] Embodiment 7: Glucose Oxidase 0.63% Albumin 0.5% Glutamic acid0.3% Potassium ferricyanide 6% Phosphate buffer solution at pH 7.092.57%

[0042] Embodiment 8: Glucose Oxidase 0.21% Dextrin 0.39% Glutamic acid0.3% Potassium ferricyanide 3.8% Phosphate buffer solution at pH 5.195.3%

[0043] As shown in FIG. 4A through FIG. 4H—the production flow charts ofthis invention, the procedure includes the following steps:

[0044] 1. Please refer to FIG. 4A. To print at least one layerconductive film 2 with screen printing technique including at least ananode and a cathode. The material of the conductive film 2 can be acarbon paste, silver plasma, mixed plasma of carbon and silver, volatilegraphite, or copper paste, which can be used independently or in pack(for example, printing the carbon paste after the silver plasma), thenheated at 40° C.˜120° C. for drying.

[0045] 2. Please refer to FIG. 4B. To print an electric insulating layer3 at least in 0.6 mm thick on the same face with the printed conductivefilm 2 by using screen printing technique, except some bare areareserved for forming the anode coupling 201, cathode coupling 211, workelectrode 200, and reference electrode 210. A circular area formed bythe work electrode 200 and the reference electrode 210 is called thearea of biological active layer.

[0046] 3. Please refer to FIG. 4C. To print a layer of the cellulosecarrier on the circular area of the biological active layer with screenprinting technique.

[0047] 4. Please refer to FIG. 4D. To dry the carrier 5 in roomtemperature (20° C.˜30° C.).

[0048] 5. Please refer to FIG. 4E. To coat the surface surrounding thecircular area of the biological active layer with glue 70 and have thenet protector 7 adhered to cover the carrier 5 as mentioned in step 3.

[0049] 6. Please refer to FIG. 4F. To drip the buffer solution 8containing the biological active substance and conductive mediator intothe carrier 5 in the circular area, where the buffer solution 8 will beabsorbed by cellulose of the carrier 5.

[0050] 7. Please refer to FIG. 4G. To dry the plate electrode madeaccording to step 6 in ambient temperature at 40° C.˜60° C. to completethe manufacture process as shown in FIG. 4-H.

[0051] From above description, it is understood that this inventionprovides a relatively speedy manufacture process for plate electrode byvirtue of coating the cellulose layer of carrier on the biologicalactive layer to adsorb the biological substance and conductive mediator,so that the work electrode and the reference electrode can be changedfrom hydrophobic into hydrophilic, and the biological substance can beprotected against impairment in subsequent manufacture process, andmoreover, the screen printing method facilitates a high productionyield.

[0052] Although, this invention has been described in terms of preferredembodiments, it is apparent that numerous variations and modificationsmay be made without departing from the true spirit and scope thereof, asset forth in the following claims.

What is claimed is:
 1. A disposable plate electrode, comprising: a planesubstrate; a conductive film disposed on one of two relative largersurfaces of said substrate; an electric insulating layer partiallycovering said conductive film for the bare portion of the latter to format least a work electrode, a reference electrode, an anode portion, anda cathode electrode, and at least, a biological active layer of saidwork electrode and reference electrode being covered by the former,wherein said biological active layer is provided with a carrier coveringsaid work electrode and reference electrode; said carrier of cellulosehas adsorbed a biological active substance and a conductive mediator,which, the biological active substance, can react with a test samplechemically or biochemically.
 2. The disposable plate electrode of claim1 , wherein said substrate can be a board of PVC, FRP (FR-4), Polyestersulfone, Bakelite, PET, PC, Glass, or ceramics (CEM-1), etc.
 3. Thedisposable plate electrode of claim 1 , wherein said carrier is ablended paste suitable for screen printing, and the composition includesmicrocrystalline cellulose, high molecule polymer, salt, and water. 4.The disposable plate electrode of claim 3 , wherein grain size of saidmicrocrystalline cellulose is 100 μm down, 10%˜40% adulterated.
 5. Thedisposable plate electrode of claim 3 , wherein said high moleculepolymer with 10%˜25% adulterated can be PVA (Polyvinyl alcohol), PVP(Polyvinyl pyrrolidone), PEG (Polyethylene glycol), or gelatin, whichmay be used in single item or blended.
 6. The disposable plate electrodeof claim 3 , wherein said salt with 1%˜5% adulterated can be Dibasicpotassium phosphate, Potassium biphosphate, or Citric acid for pH valueadjustment in a proper range of pH 4.5˜pH 9.0 and can serve as a buffersolution.
 7. The disposable plate electrode of claim 3 , wherein saidwater must be pure water undergone at least one time distillation. 8.The disposable plate electrode of claim 1 , wherein said biologicalsubstance comprising a immobilized or unimmobilized enzyme (such asglucose oxidase), antigen, antibody, microbe cell, animal or plant cell,animal or plant tissue, etc, which possess biological cognizablespecialties, being used to contact the test sample (biological tissuesuch as blood) for chemical or biochemical reaction.
 9. The disposableplate electrode of claim 1 , wherein said conductive mediator meansPotassium ferricyanide and the like with 2%˜10% adulterated, which isused to receive electrons released after reaction of an enzyme and thetest sample, and the electrons will be transmitted via an electrodeconductor to a sensor to be transferred into sample concentration. 10.The disposable plate electrode of claim 8 , wherein said biologicalactive substance must be blended with said conductive mediator for use;and composition thereof comprises enzyme, enzyme protector, conductivemediator, and phosphate buffer solution.
 11. The disposable plateelectrode of claim 10 , wherein said enzyme (such as glucose oxidase) isadulterated at 200 U˜1200 U/ml.
 12. The disposable plate electrode ofclaim 10 , wherein said enzyme protector adulterated at 0.1%˜1% can bealbumin, dextrin, dextran, or amino acid, and can be used in a singleitem independently or in blended.
 13. The disposable plate electrode ofclaim 10 , wherein said conductive mediator adulterated at 2%˜10% meansPotassium ferricyanide.
 14. The disposable plate electrode of claim 10 ,wherein a proper pH value of said phosphate buffer solution is 4.8˜7.5.15. A manufacture method for forming a biological active layer to adisposable plate electrode, comprising: using screen printing techniqueto print a conductive film on one of two relatively larger surfaces of asubstrate including at least an anode electrode and a cathode electrode,and drying it under ambient temperature 40° C.˜120° C.; using screenprinting technique to print an electric insulating layer overlappingsaid conductive film except some reserved bare portion of the latterwhich is to be used forming an anode coupling, a cathode coupling, awork electrode, a reference electrode, and a circular area (an area ofbiological active layer) formed by said work and reference electrodes;using screen printing technique to print a layer of cellulose carrier onsaid circular area, and drying it under room temperature (20° C.˜30°C.); gluing the peripheral portion of said electric insulating layeraround the circular area of said biological active layer, and theretosticking a net protector to cover the circular area of said biologicalactive layer; dripping water solution of biological active substance andconductive mediator to surface of said carrier, and drying it underambient temperature 40° C.˜60° C. to complete the disposable plateelectrode.